Preclinical development of complex processing systems for ex vivo culture-expanded lymphohematopoietic cells, with subsequent immunologic and/or genetic manipulation, have been carried out in collaboration with a number of NIH institute investigators. oPreparation of CD8-depleted, culture-expanded lymphocytes: In collaboration with NIAID and NCHGR, this 10-day process that uses OKT3 + IL2 for T-cell culture/expansion has been applied to a clinical trial of HIV gene therapy in the syngeneic twin model, and the first phase of the clinical trial was completed in June 1998. Several methodologic innovations have been evaluated over the past 2 years, including evaluation of new (non-Neo) vectors, T-cell culture/expansion with CD3/CD28 beads (June method?see below) to enhance T-cell function and longevity, and use of fibronectin-coated bags for improved gene transduction (see below). The clinical trial is on hold pending regulatory approvals. oPreparation of allogeneic donor lymphocytes selectively depleted for alloreactive T-cells: This process is being developed, in collaboration with NHLBI, for application to clinical allogeneic hematopoietic transplantation, especially in the HLA-mismatched setting. We had previously developed a reliable method (using OKT3 + IL2) for expansion of selected T-cells from patients with CML, resulting in a product that has minimal contamination with leukemia cells and is capable of stimulating a third-party (donor) immune response to recipient-specific antigens. To date, five clinical scale expansions have been done, and the expansion characteristics appear to vary with the severity of the patient's leukemia. This project was re-focused in FY 2000 by a Bench-to-Bedside award, which dedicated a technologist to the work. We plan to develop and scale up a method based on depletion of CD25+ cells following an allogeneic MLR, in order to deplete alloreactive donor cells from a lymphocyte graft. A clinical trial is planned for FY 2001. oT lymphocyte culture/expansion in Aastrom Bioreactor: Over the past 2 years, studies were done with the goal of adapting the T-cell culture process described above to the Aastrom Replicell bioreactor system. Six culture-expansion procedures have been done using a manual version of the Replicell system; this developmental demonstrated the feasibility of using this system to expand lymphocytes. A manuscript is in preparation. oNovel methods for culture/expansion of lymphocytes: We have adapted the T-cell culture/expansion method of Carl June (Univ. of Pennsylvania) to NIH clinical protocol needs. This method uses an immunomagnetic bead that is coated with both OKT3 and CD28 to select and expand the T-cell population with the goal of eliminating anergy or apoptosis that have been shown to occur with the traditional (OKT3 + IL2) expansion method. Another immunomagnetic method that combines depletion of CD8 (T-suppressor) and CD20 (B) cells prior to culture has been developed for application to the HIV syngeneic twin studies and NCI studies of post-transplantation immunotherapy. In the latter application, the two different methods have been applied to the generation of Th2 lymphocytes that will be co-cultured with dendritic cells to effect a reduction in graft-versus-host-disease; this process will go into clinical trial in FY 2001. oFibronectin transduction: A method for improved gene transduction using fibronectin-coated bags was developed and incorporated into the clinical trial of gene therapy for chronic granulomatous disease. This method may be incorporated into the HIV syngeneic twin gene therapy studies that involve transduction of lymphocytes, but will require validation in that system.